Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin, which is an antitoxic substance produced by a plant tissue in response to external toxicity and found in a wide variety of dietary sources including grapes, plums, and peanuts. It exhibits beneficial effects including anti-oxidant, anti-inflammatory, cardioprotective, and anti-tumor activities (Kundu and Surh, 2008; Pirola and Fröjdö, 2008; Athar, et al., 2007). Currently, numerous preclinical findings suggest resveratrol as a promising nature's arsenal for cancer prevention and treatment. As a potential anti-cancer agent, resveratrol has been shown to inhibit or retard the growth of various cancer cells in culture and implanted tumors in vivo. The biological activities of resveratrol are found to relate to its ability in modulating various targets and signaling pathway.
Piceatannol (3,5,3′,4′-tetrahydroxystilbene) is a polyphenol found in grapes and other plants. It is known as a protein kinase inhibitor that exerts immunosuppressive and antitumorigenic activities on several cell lines, and has been shown to exert various pharmacological effects on immune and cancer cells (Kim et al, 2008b and references therein). In humans, piceatannol is produced as a major metabolite of resveratrol by CYP1B1 and CYP1A2 (Potter et al., 2002; Piver et al., 2004). In addition, the metabolism of trans-resveratrol into two major metabolites, piceatannol (3,5,3′,4′-tetrahydroxystilbene) and another tetrahydroxystilbene, was catalyzed by recombinant human CYP1A1, CYP1A2 and CYP1B1 (Piver et al., 2004).

Cytochrome P450 enzymes (P450s or CYPs) constitute a large family of enzymes that are remarkably diverse oxygenation catalysts found throughout nature, from archaea to humans (available on the internet at drnelson.utmem.edu/CytochromeP450.html). Because of their catalytic diversity and broad substrate range, P450s are attractive as biocatalysts in the production of fine chemicals, including pharmaceuticals (Guengerich 2002; Urlacher et al., 2006; Yun et al., 2007; Lamb et al., 2007). In spite of the potential use of mammalian P450s in various biotechnology fields, they are not suitable as biocatalysts because of their low stability, catalytic activity, and availability.
If a metabolite, such as piceatannol, has a biological activity, direct administration of the metabolite into a living body may be beneficial. However, large quantities of the metabolite need to be produced. If pro-drugs are converted to biologically ‘active metabolites’ by human liver P450s during the drug development process (Johnson et al., 2004), large quantities of the pure metabolites are required to understand the drug's efficacy, toxic effect, and pharmacokinetics.
The pure metabolites may be difficult to synthesize. An alternative to chemical synthesis is to use P450s to generate the metabolites of drugs or drug candidates. Hepatic microsomes can be a source of human P450s, but their limited availability make their use in preparative-scale metabolite synthesis impractical. Some human enzymes can also be obtained by expression of recombinant hosts. Metabolite preparation has been demonstrated using human P450s expressed in Escherichia coli and in insect cells (Parikh et al., 1997; Rushmore et al., 2000; Vail et al., 2005), but these systems are costly and have low productivities due to limited stabilities and slow reaction rates (usually <5 min−1 (Guengerich et al., 1996)). An alternative approach to preparing the human metabolites is to use an engineered bacterial P450 that has the appropriate specificity.
The P450 BM3 (CYP102A1) from Bacillus megaterium has strong similarity to eukaryotic members of the CYP4A (fatty acid hydroxylase) family. It was shown that engineered CYP102A1 mutants could oxidize several human P450 substrates to produce the authentic metabolites with higher activities (Kim et al., 2008; Otey et al., 2005; Yun et al., 2007 and references therein). Furthermore, CYP102A1 is a versatile monooxygenase with a demonstrated ability to work on a diversity of substrates (Bernhardt et al., 2006, Di Nardo et al., 2007).
Recently, wild-type CYP102A1 has been engineered to oxidize compounds showing little or no structural similarity to its natural substrate fatty acids (Lamb et al., 2007). The compounds include testosterone, several drug-like molecules, and polycyclic aromatic hydrocarbons (PAHs), which are known substrates of human P450 enzymes (Carmichael et al., 2001; van Vugt-Lussenburg et al., 2006). However, there has been no research on whether resveratrol can be used as a substrate. A set of CYP102A1 mutants was shown to generate larger quantities of the authentic human metabolites of drugs, which may be difficult to synthesize (Otey et al., 2005). An alternative approach to preparing the metabolites is to use engineered CYP102A1 enzymes with desired properties.
Based on the scientific literature, several amino acid residues in CYP102A1 were mutated to generate mutant enzymes showing increased activity toward human P450 substrates (Yun et al., 2007). Very recently, it was reported that some selected mutations enabled the CYP102A1 enzyme to catalyze O-deethylation and 3-hydroxylation of 7-ethoxycoumarin, which are the same reactions catalyzed by human P450s (Kim et al., 2008a).
There are several patent applications related to piceatannol. That is, a composition for antihypertensive effects comprising a Rhei Rhizome extract or active compounds isolated therefrom is disclosed in Korean Patent Application No. 10-2005-0126879, and a cosmetic composition containing piceatannol and vitamin A is disclosed in Korean Patent Application No. 10-2007-0025087. However, a patent application related to a method of preparing piceatannol has not been filed. While a method of chemically synthesizing resveratrol and piceatannol is disclosed in WO2008012321, a method of biologically preparing resveratrol and piceatannol has not been disclosed.
All cited references are incorporated herein by reference in their entireties. The information disclosed herein is intended to assist the understanding of technical backgrounds of the present invention, and cannot be prior art.